2.7.8. Real‐time polymerase chain reaction (RT‐PCR) assay
RT‐PCR was used to study the effect of prepared scaffolds and PEMF on the osteogenic gene expression ,including collagen type 1 (Col1), Runt-related transcription factor 2 (Runx2), and Osteocalcin (OC). At first, total mRNA was extracted using Qiazol reagent after 14 days of culturing MSCs. Afterward, cDNA was synthesized by MLV reverse transcriptase (RT) ,and random hexamer according to the manufacturer’s instructions (Fermentas). The rate of gene expression was quantified by an RT-PCR analyzer (Corbett, Australia) and Rotor-Gene software was used to quantify the gene expression level. In this study, the expression of the target gene was evaluated via the relative quantification model compared to β2M control as a housekeeping gene. Relative gene expression was calculated by ∆∆Ct method. The primer sequences of corresponding genes are presented in Table 1.
Results and Discussion
In this study, we synthesized iron oxide nanoparticles showing superparamagnetic properties. These nanoparticles highly tend to aggregate ,which increases the overall size of the nanoparticles and consequently precipitate them. Thus, surface modification seems necessary to stabilize SPIONs and improve their biocompatibility (Gupta & Gupta, 2005; Hamley, 2003; Ito et al., 2005). PAMAM dendrimer was chosen as an appropriate surface modification because of its high water solubility, biocompatibility, antimicrobial properties, and positively charged amine groups (Chawla, Shetty, Goyal, Rathore, & Sharma, 2018; Fox, Richardson, & Briscoe, 2018).
The size and morphology of G3-SPIONs have been analyzed using the TEM instrument and shown in Fig. 2a. The prepared G3-SPIONs had spherical shape with the uniform size distribution. The average diameter of nanoparticles was measured by the ImageJ software (NIH, Bethesda, MD) and estimated to be 17.95 ± 3.57 nm. The three-dimensional structure of dendrimers endues SPIONs steric stabilization, and thus well-dispersity (Yen, Lien, Chung, & Yeh, 2017).
G3-SPIONs were also characterized by DLS ,which measures the hydrodynamic diameter of the MNPs in their dispersion state (Fig. 2b). The result showed that the average size of the nanoparticles was 28.91 nm ,which as expected, was more significant than the size obtained by transmission electron microscopy (Souza, Ciminelli, & Mohallem, 2016). The surface charge of G3-SPIONs ,which is a crucial parameter for the electrostatic interaction between cells and the surface of the material was assessed by zeta potential measurement. The zeta potential of G3-SPIONs was 22.7 mV due to amine groups in PAMAM dendrimer with a positive charge (Fig. 2c). Since some of G3-SPIONs may be exposed on the nanofiber surface, the positive charge can increase the cell attachment on the scaffold’s surface and consequently improve cell-substrate interactions.
SPIONs functionalization and coating by APTES and PAMAM dendrimers, respectively, on the surface of SPIONs were approved by FT-IR spectroscopy, as shown in Fig. 3a. The characteristic absorption band at 586 cm-1 is attributed to the stretching bond of the Fe–O which confirms the presence of Fe3O4 nanoparticles. The peak at 1021 cm-1 is assigned to the stretching vibration of the Si-O-Fe bond, proving the presence of SiO2 shell on the surface of SPIONs. The peaks around 2866 cm-1 and 2912 cm-1 are due to the existence of stretching vibration of CH2 in the aminopropyl group which confirms the binding of APTES molecules. The broad peak at 3430 cm-1 is due to the stretching mode of the NH2 group and the band at 1634 cm-1exhibits a stretching vibration of –CO–NH– group, representing the binding of PAMAM dendrimers on the surface of SPIONs. It can be observed that increasing the order of PAMAM generation increases the intensity of these peaks.
The magnetization property of SPIONs ,and G3-SPIONs was measured using VSM at room temperature . As shown in Fig. 3b, the negligible remanence (Mr) and zero coercivity (HC) of the magnetic nanoparticle confirm the superparamagnetic behavior of the samples ,which prevents the formation of a stable magnetic state and enables them to re-disperse rapidly in ,the absence of magnetic field. compared with the maximum magnetization (MS) value of 68.28 emu/g for SPIONs, the Ms value for G3-SPIONs decreases to 57.75 emu/g due to the linkage of non-magnetic PAMAM dendrimer on ,the surface of SPIONs. Non- magnetic layer of PAMAM inevitably lead to an unavoidable decrease in saturation magnetization. It can result from a decrease in the strength of the exchange interaction between oxygen and iron atoms (Uzun et al., 2010). According to Taghavi et al., the VSM result of bare MNPs analyzed at 37 ⸰C was obtained as 48.8 emu/g (POURIANAZAR, 2016) Zhu et al. have prepared Poly peptide dendrimers through the ligand-exchange method synthesized on MNPs. The saturation magnetization for G3(Lysine) and G3(Glutamine) were reported 42.9 and 41.6, respectively. According to recent studies, the saturation magnetization is related to the particle size, directly. The reason for the decrease in saturation magnetization arises from the increase in non-magnetic surface coverage of each particle G3(Lys) and G3(Glu) as ligand-exchange (Zhu et al., 2011). Considering the mentioned studies, it could be concluded that the saturation magnetization value of the SPION and G3-SPION is comparable with that obtained in the previous studies.
The G3-SPIONs was embedded into the PCL nanofibers to be used as a scaffold in the osteogenesis differentiation. The SEM micrographs of PCL and G3-SPION-PCL nanofibrous scaffolds are presented in Fig. 4, to analyze the diameter and morphology of the prepared scaffold. As shown, the nanofibrous scaffolds have a cylindrical shape without any structural imperfection resulting from the proper selection of electrospinning parameters. The average fiber diameter of the PCL and G3-SPION-PCL nanofibers obtained 866±310 and 495±144, respectively. It was observed that the presence of G3-SPIONs in the scaffold has reduced the size and distribution of fiber diameter ,which is justifiable by higher conductivity of the polymer solution in the presence of G3-SPIONs. High porosity and nanostructure property of the scaffolds endue a high potential for cell attachment ,which can be used in tissue engineering. Although the suitable average diameter of electrospun PCL nanofibers for bone tissue engineering was reported in the wide range, from 20 to 5000 nm, the average fiber diameter of the prepared nanocomposite fibers was in the range of bone tissue engineering applications, as confirmed by other studies (Jang, Castano, & Kim, 2009; Ren, Wang, Sun, Yue, & Zhang, 2017).
The MTT assay was used to investigate the proliferation rate and viability of ADMSCs on electrospun PCL and G3-SPION-PCL scaffolds on days 1, 3, 7, and 14. As shown in Fig. 5a, cell growth, and proliferation have an increasing trend for all groups, indicating the biosafety of the prepared scaffolds. As can be observed, there was no statistically significant difference between the OD value of the cells cultured on the PCL and G3-SPION-PCL on day 1. However, the difference has been increased until the significant discrepancy has been observed on day 14, indicating a higher rate of proliferation and growth on G3-SPION-PCL compared with PCL scaffold. The better cell growth on G3-SPION-PCL can be attributed to G3-SPIONs nanoparticles ,which endue more hydrophilicity and positive surface charge to the prepared scaffold, leading to the improved cell growth and proliferation. It is noticeable that the porous structure of the scaffolds causes the migration of some seeded cells from the porous nanofibrous scaffold to the TCP plate, resulting in lower OD value of the scaffolds compared with TCP.
The potential of PCL and G3-SPION-PCL nanofibrous scaffolds on cell attachment and ADMSCs proliferation rate was also evaluated by DAPI staining on days 1 and 14. As shown in Fig. 5b, the scaffold containing G3-SPIONs showed enhanced cell attachment ,and growth compared to the neat scaffold, PCL. The MTT assay indicated that the G3-SPION-PCL scaffold was more compatible than PCL and according to the images of DAPI staining the number of attached cells to G3-SPION-PCL was much more than PCL. It can be concluded that the presence of G3-SPIONs decreases the fiber diameter, increases the surface to volume ratio,and affect significantly on the cell attachment. Overall, surface topography, hydrophilicity, and positively charged PAMAM dendrimers ,which were partially exposed on the scaffold surface play a vital role in the interaction of cells to the scaffold (Gloria et al., 2013).
An electromagnetic bioreactor was used to induced EMF and enhance the osteogenesis of ADMSCs mediated by incorporated SPIONs. Many studies demonstrated that EMF has a positive effect on the osteoblast and adipocyte differentiation of mesenchymal stem cells (Wang, Wu, Yang, & Song, 2016). Possible effects of scaffold modification via incorporation of the dendrimerized SPIONs, in the presence of biochemical (OM), and biophysical signaling factors (PEMF) on osteogenesis potential of ADMSCs was evaluated by monitoring the expression of the osteogenic marker on days 7 and 14 after cell seeding.
ALP activity is known as an early marker of osteogenic differentiation. The research studies have indicated that the ALP enzyme plays an essential role in the initiation of mineralization (Miron & Zhang, 2012). As shown in Fig. 6a, the presence of SPIONs, OM, and PEMF resulted in a significant difference in ALP activity between G3-SPION-PCL/OM/PEMF and the other group. The presence of both biochemical and biophysical factors increased the ALP activity ,which is in line with the previous studies reported on the positive effect of electromagnetic waves on the osteogenic differentiation (Xia et al., 2018). Dankova et al. (Daňková et al., 2015) demonstrated that the PCL-MNPs scaffolds increased the ALP activity significantly in cells cultivated on the PCL-MNPs on day 7 (OD around 0.2). Here, higher ALP activity could be due to dendrimers coated MNPs, which significantly promote adhesion density and osteoblast proliferation via 3D structure and considerable positive charge. The calcium deposition value, as the late marker of osteogenic differentiation, was evaluated quantitatively by calcium content assay, Fig. 6b. As shown, there were no significant differences between the amount of calcium deposition in TCP and PCL scaffold. While the calcium content of G3-SPION-PCL/OM, G3-SPION-PCL/PEMF, and G3-SPION-PCL/OM/PEMF is significantly higher than TCP and PCL. Also, G3-SPION-PCL/OM/PEMF significantly showed higher calcium content than the other groups, confirming the synergistic effect of OM and PEMF in the osteogenic differentiation of ADMSCs. Since the G3-SPION-PCL/OM/PEMF group demonstrated the best ALP activity ,as well as calcium deposition in the osteogenic differentiation, SEM analysis of this sample was performed to investigate the cell adhesion, morphological features, , and shape of the cells on days 1 and 14. As shown in Fig. 6c, no calcium precipitation was observed on the first day, but upon differentiation on day 14 led to a higher level of calcium deposition. Dendritic architecture introduced benefits over linear polymers with the aim of in vitro bone cell growth and adhesion (Joshi & Grinstaff, 2008). Also, it was observed that the morphology of hMSCs showed a significant change from a fibroblastic shape into a polygonal shape during osteodifferentiation (Erices, Conget, Rojas, & Minguell, 2002). This study confirms that the rational design of dendritic architectures can result in the fabrication of new scaffolds with improved properties. The effect of PEMF and OM on the expression level of osteogenic transcription factors was evaluated on day 14. As shown in Fig. 7, the ADMSCs seeded on the G3-SPION-PCL scaffold in the presence of OM and exposure to PEMF expressed the highest level of Osteocalcin and Runx2. Osteocalcin, as the most plentiful non-collagenous bone matrix protein, is mainly expressed by osteoblasts and serves a specific marker for the late stages of bone differentiation. Whenever Osteocalcin is overexpressed, the mineral species maturation will be accelerated and modulates osteogenic differentiation of MSCs (Tsao et al., 2017). The superposition of OM and PEMF resulted in significant expression of this gene by ADMSC cultured on the G3-SPION-PCL scaffold.
Collagen type I (Col 1) is an early marker of bone differentiation and the most abundant protein in the bone matrix. The result (Fig. 7) demonstrated overexpression of Col 1 by the cells cultured on the G3-SPION-PCL scaffold under the only PEMF exposure. Interestingly, this result showed that PEMF exposure accelerates early-stage osteogenic differentiation. However, in the combination with OM and PEMF exposure, Col 1 expression did not show such elevation. Recent researches have proved that PEMFs improve osteogenic differentiation of MSCs by stimulating the mRNAs expression of osteogenic related genes such as RunX2, ALP, and BMP2 (Chen et al., 2019). Taking together, it could be concluded that the presence of G3-SPIONs in the PCL scaffold along with simultaneously PEMF exposure and OM increases the differentiation rate of ADMSCs to osteogenic linage and can be a suitable candidate in bone tissue engineering.
Conclusion
In this study, SPIONs were synthesized by the co-precipitation method and modified by G3-PAMAM dendrimer using a divergent method. The nanoparticles were incorporated into the PCL scaffold by electrospinning to produce a novel magnetic scaffold. The influence of G3-SPIONs incorporation on the properties of the magnetic electrospun nanofibers were investigated. MTT assay and DAPI staining confirmed enhancement in growth and proliferation of stem cells seeded on magnetic G3-SPION-PCL compared to the pure PCL. After that, the osteogenic differentiation of the magnetic electrospun scaffolds under biochemical (OM) and biophysical (PEMF) stimulations were investigated ,and analyzed by calcium content assay, ALP activity and real-time PCR. Results interestingly confirmed the superposition effect of G3-SPIONs and PEMF to increase osteogenic differentiation of ADMSCs.
In conclusion, this study suggests that G3-SPION incorporated PCL nanofibers could be considered as a potential magnetic scaffold for bone tissue regeneration applications.